The molecular mechanisms of aging and degenerative diseases are not completely understood, but it is known that the level of oxidative protein damage in mitochondria increases markedly with age and is high in patients with rapid-aging syndrome as well as those with degenerative diseases such as Alzheimer's. Carbonylation is a common oxidative modification and recent studies indicate that it is selective and concentrated in a subset of cellular proteins. However, little is known about the specific sites of carbonylation in these proteins. This information is critical because it provides a platform for understanding how reactive oxygen and carbonyl species broadly alter protein structure and leave footprints, which can potentially act as markers for the disease states and aging processes. Moreover, it is a key to assessing the modes of action of therapeutics aimed at ameliorating the effects of oxidative damage to mitochondria. The research team is well suited for the proposed studies and brings together expertise in mass spectrometry and organic reaction mechanisms (Gronert), genetics (Grotewiel and Bettinger), Drosophila melanogaster (Grotewiel), and C. elegans (Bettinger). In the present application, we will identify carbonylation sites in proteins subjected to oxidative stress, both in model protein systems as well as in model organisms. Our working hypothesis is that protein carbonylation is localized at vulnerable structural motifs and depends on the nature of the oxidant. To test this hypothesis, model proteins will be subjected to oxidative stress with typical cellular oxidants or oxidation products (i.e., unsaturated aldehydes) and analyzed with a sensitive mass spectrometric approach. In addition, we will use a similar approach to examine mitochondrial protein from Drosophila melanogaster and C. elegans. Here, the effect of aging and induced oxidative stress on the level and nature of oxidative protein damage will be assessed. The work will yield the following outcomes: (a) carbonylation maps and structure/reactivity relationships in model proteins subjected to oxidative stress, (b) background oxidation sites in the mitochondrial protein of two model organisms, (c) direct measures of the effect of in vivo oxidative stress on protein carbonylation, and (d) determination of the impact of oxidative stress on protein levels in the electron transport chain. The rationale for the work is that by identifying the specific sites of carbonyl-related protein modifications, we can pinpoint ways in which protein structures are altered and discover patterns in oxidative modifications that correlate with the form of oxidative stress. This information is critical for understanding the role of protein oxidation in mitochondrial dysfunction and for the development of biomarkers based on protein oxidation as well as the assessment of potential therapeutics.